Identification and Analysis of Three Myristylated Vaccinia Virus Late

JOURNAL OF VIROLOGY,0022-538X/97/$04.0010

July 1997, p. 5218–5226 Vol. 71, No. 7

Copyright © 1997, American Society for Microbiology

Identification and Analysis of Three MyristylatedVaccinia Virus Late Proteins

KAREN H. MARTIN, DOUGLAS W. GROSENBACH, CHRISTINE A. FRANKE,AND DENNIS E. HRUBY*

Department of Microbiology, Center for Gene Research and Biotechnology,Oregon State University, Corvallis, Oregon 97331-3804

Received 6 January 1997/Accepted 19 March 1997

Previous studies have shown that at least three vaccinia virus (VV) late proteins (with apparent molecularasses of 37, 35, and 25 kDa) label with myristic acid. Time course labeling of VV-infected cells with [3H]myristic acidreveals at least three additional putative myristylproteins, with apparent molecular masses of 92, 17, and 14kDa. The 25-kDa protein has previously been identified as that encoded by the L1R open reading frame, leavingthe identities of the remaining proteins to be determined. Sequence analysis led to the preliminary identifi-cation of the 37-, 35-, and 17-kDa proteins as G9R, A16L, and E7R, respectively. Using synthetic oligonucle-otides and PCR techniques, each of these open reading frames was amplified by using VV DNA as a templateand then cloned individually into expression vectors behind T7 promoters. These plasmid constructs were thentranscribed in vitro, and the resulting mRNAs were translated in wheat germ extracts and radiolabeled witheither [35S]methionine or [3H]myristic acid. Each wild-type polypeptide was labeled with [35S]methionine or[3H]myristic acid in the translation reactions, while mutants containing an alanine in place of glycine at theN terminus were labeled only with [35S]methionine, not with myristic acid. This result provided strong evi-dence that the open reading frames had been correctly identified and that each protein is myristylated on aglycine residue adjacent to the initiating methionine. Subcellular fractionations of VV-infected cells suggestedthat A16L and E7R are soluble, in contrast to L1R, which is a membrane-associated protein.

Vaccinia virus (VV) is a large double-stranded DNA virusthat replicates in the cytoplasm of infected cells. It encodesmost, if not all, of its own enzymes and cofactors required fortranscription and DNA replication while relying on the hostcell machinery for translation of viral mRNAs (37). Like manyother eukaryotic viruses, VV utilizes many of the same strat-egies as the host cell for trafficking proteins to their properlocations within the cell. Posttranslational modifications suchas proteolysis, phosphorylation, glycosylation, and fatty acyla-tion all participate in this process (55).

The two predominant types of fatty acylation known to occuron viral proteins are palmitylation and myristylation (26). Inboth cases, a saturated fatty acid is transferred to the protein,generally serving to target the modified protein to a membraneor to stabilize protein-protein interactions (28). Myristic acid isa 14-carbon fatty acid that is cotranslationally transferred tothe penultimate glycine residue found within the consensussequence MGXXX(S/T/A/C/N) (where X is any amino acid) atthe amino terminus of target proteins (13). The enzyme re-sponsible for the transfer of myristic acid from myristoyl co-enzyme A to the target protein is N-myristyltransferase. Inaddition, a second class of myristylproteins has recently beenrecognized. Two examples of the latter class are the tumornecrosis factor alpha and interleukin-1a precursors, whichhave both been shown to be myristylated on internal lysinedoublets (49, 50). Nothing is yet known about the enzymeresponsible for this modification or its substrate specificity.Among viral myristylproteins, only N-terminally myristylatedproteins have been characterized, although the major capsidprotein of the Paramecium bursaria chlorella virus 1 algal virusappears to be a candidate for internal myristylation (41).

Many viruses express proteins that are modified by myristicacid. The majority of viral myristylproteins are components ofthe capsid or envelope. The roles that these proteins play in thevirus life cycle are varied. With few exceptions, inhibition ofmyristylation is detrimental to the virus. For example, themajor picornavirus myristylprotein is VP4. Myristylation of thisprotein is essential for assembly of infectious virions (8), andevidence suggests that it may also play a role in the uncoatingprocess following entry into the host cell (36). Likewise, myris-tylation is required for the translocation of human immunode-ficiency virus Gag protein to the plasma membrane, wherevirion assembly and budding occur (5, 19, 21). Transformationof cells by Rous sarcoma virus is also dependent on myristyla-tion. The myristylprotein, pp60v-src, is a membrane-associatedtyrosine kinase (6). If myristylation is blocked, the virus is nolonger able to transform cells (45).

The best-characterized VV myristylprotein is encoded by theL1R open reading frame (ORF). This protein is approximately25 kDa in size, is myristylated at the N terminus within thestandard consensus sequence, and has two predicted trans-membrane domains (17, 42). Previous work has shown thatL1R localizes to the inner membrane of mature virions and isrequired for virus growth in tissue culture. Use of an induciblerecombinant virus demonstrated that absence of L1R results ina shutdown of morphogenesis at an early stage, preventingformation of mature intracellular virions (43). Studies usingmonoclonal antibodies also suggest a role for L1R in penetra-tion of adsorbed virus and in acid-induced fusion of infectedcells (57).

In addition to L1R, there are at least five other VV myri-stylproteins. This is an unusually large number of myristylpro-teins to be expressed by one virus; however, due to the nearly200 predicted ORFs found in the viral genome (20) and themultiple forms of viral particles (47, 48), it is perhaps notsurprising. We report here on the identities of three of these

* Corresponding author. Phone: (541) 737-1849. Fax: (541) 737-2440. E-mail: [email protected].

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additional VV myristylproteins, A16L, E7R, and G9R. Surpris-ingly, A16L and E7R were both found to be soluble, in starkcontrast to L1R. Since these proteins are not associated withmembranes, they either are most likely involved in virion as-sembly or are soluble enzymes within the core of virus parti-cles.

MATERIALS AND METHODS

Cells and virus. BSC-40 (African green monkey kidney) cells and RK13(rabbit kidney) cells were maintained in modified Eagle’s medium (MEM-E;Sigma Chemical Co., St. Louis, Mo.) supplemented with 10% heat-inactivatedfetal bovine serum (Whittaker M.A. Bioproducts, Inc., Walkersville, Md.), 2 mML-glutamine, and 10 mg of gentamicin per ml. The Western Reserve (WR),Copenhagen (CH), and IHD-J strains of VV were purified as previously de-scribed (27).

Subcellular fractionation. Confluent monolayers of BSC-40 cells (8.9 3 106

cells) were infected with IHD-J virus at a multiplicity of infection (MOI) of 10PFU/cell. After a 1-h adsorption, the inoculum was replaced with MEM-Econtaining 5% heat-inactivated fetal bovine serum. The infected cells were in-cubated at 37°C with 5% carbon dioxide for 24 h. Cells were harvested in culturemedium and were pelleted by centrifugation at 1,300 rpm for 10 min at 4°C. Thecell pellet was resuspended in 5 ml of hypotonic buffer (20 mM HEPES, 5 mMKCl, 1 mM MgCl2, 150 mM NaCl). Cells were swollen on ice for 10 min and thenDounce homogenized. One milliliter was removed (total cell extract [TCE]). Theremainder was centrifuged at 1,300 rpm for 10 min at 4°C. The pellet (nuclearpellet [NP]) was brought to 4 ml in hypotonic buffer. The supernatant (post-nuclear supernatant [PNS]) was separated into 1-ml fractions, two of which weremicrocentrifuged at 15,000 rpm for 30 min at 4°C. The resulting pellets (P15)were brought to 1 ml in hypotonic buffer; the supernatants (S15) were combined,brought to 4 ml with hypotonic buffer, and centrifuged at 33,000 rpm for 1 h at4°C. The pellet of the 100,000 3 g spin (P100) was brought to 1 ml in hypotonicbuffer; the supernatant (S100) was adjusted to a final concentration of 10%trichloroacetic acid and ultracentrifuged at 18,000 rpm for 30 min at 4°C. Thepellet (precipitated S100) was resuspended in 600 ml of 1 M Tris-HCl (pH 11.0)to neutralize the acid and then divided into two 300-ml fractions. All othersubcellular fractions were brought up to 10% trichloroacetic acid and microcen-trifuged at 15,000 rpm for 30 min at 4°C. The protein pellets were resuspendedin 300 ml of 1 M Tris-HCl (pH 11.0) except for P100, which was treated the sameas the S100 fraction. The pH of each fraction was adjusted to 7.0 with sodiumacetate. The fractions were resuspended by sonication, vortexing, and one freeze-thaw cycle.

Radiolabeling of VV-infected cells. Monolayers of BSC-40 cells were infectedwith either the WR or CH strain of VV as described below. Cells were labeledwith [9,10-3H]myristic acid (125 mCi/ml, 22.4 Ci/mmol; New England Nuclear)from 3.5 to 10 h postinfection (p.i.). Myristic acid was dried under nitrogen gasflow and resuspended in dimethyl sulfoxide (DMSO; 10 ml per plate of cells) withbrief sonication. MEM-E plus 5% fetal bovine serum was added (1 ml per plateof cells). Infected cell monolayers were washed with phosphate-buffered saline(137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4–7H2O, 1.4 mM KH2PO4, 1 mMMgCl2; PBS-M), scraped off the plates, and centrifuged at 2,000 rpm for 5 minto pellet the cells. The cell pellets were resuspended in 200 ml of lysis buffer (50mM Tris-Cl [pH 8], 1 mM EDTA, 100 mM NaCl, 1 mg of aprotinin per ml, 100mg of phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40) by vortexing. Followingtransfer to a 1.5-ml microcentrifuge tube, the samples were microcentrifuged at3,000 rpm for 2 min. The supernatant was aliquoted into 25-ml fractions andstored at 270°C. A fraction of each lysate was boiled in Laemmli sample buffer(50 mM Tris [pH 6.8], 1% [wt/vol] sodium dodecyl sulfate [SDS], 0.1% [vol/vol]2-mercaptoethanol, 1% [vol/vol] glycerol [33]) for 3 min. Proteins were resolvedby discontinuous polyacrylamide gel electrophoresis (PAGE) on an SDS–12%polyacrylamide gel. Proteins were visualized by fluorography with 22.2% 2,5-diphenyloxazole (PPO) in DMSO (4). Gels were dried and exposed to KodakBiomax MR film at 270°C.

Time course label with myristic acid. Cells infected with the WR strain of VVwere radiolabeled with [9,10-3H]myristic acid (125 mCi/ml, 22.4 Ci/mmol; NewEngland Nuclear) as described above from 0 to 4, 4 to 8, 8 to 12, 12 to 16, and16 to 24 h p.i. Cells were aspirated and then harvested in 0.1% SDS, andbenzonase endonuclease (25 U/ml; VWR Scientific) was added to shear DNA.The lysates were brought to 1% SDS, and aliquots were boiled for 3 min inLaemmli sample buffer. Proteins were resolved by discontinuous PAGE on a10% tricine gel (46) and visualized by fluorography with 22.2% PPO in DMSO(4). Gels were dried and exposed to Kodak Biomax MR film at 270°C.

Immunoblotting. Infected cells were harvested as described above, and TCEswere analyzed by SDS-PAGE. Proteins were electrotransferred from gels tonitrocellulose filters in Towbin transfer buffer (25 mM Tris [pH 8]), 192 mMglycine, 20% [vol/vol] methanol) for 30 min at 25 V at 4°C. Filters were washedonce in Tris-buffered saline (TBS; 20 mM Tris, 500 mM NaCl [pH 7.5]) andblocked for 2 h at room temperature with 3% (wt/vol) gelatin (Bio-Rad, Rich-mond, Calif.) in TBS. The filters were washed with TTBS (0.05% Tween 20 inTBS) three times, followed by binding with polyclonal antisera (as specified) inantibody buffer (1% gelatin in TTBS) overnight at room temperature. Filters

were washed three times with TTBS and then bound with alkaline phosphatase-conjugated goat anti-rabbit antibody (dilution of 1:2,000; Bio-Rad) in antibodybuffer for 1 h at room temperature. After three washes with TTBS and one withTBS, the filters were developed in p-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (Bio-Rad) in carbonate buffer (100mM NaHCO3, 1.0 mM MgCl2 [pH 9.8]).

Preparation of intracellular mature virus (IMV) and extracellular envelopedvirus (EEV). Five 150-mm-diameter plates of confluent RK13 cells were infectedwith IHD-J virus at an MOI of 5 PFU/cell. After a 24-h incubation at 37°C (5%CO2), the infected cells were harvested and transferred to a 50-ml conical tube.The cells were allowed to swell by incubating on ice for 15 min and thensonicated with a probe sonicator for 15 pulses, 3.75 duty cycles. The lysate wascentrifuged at 2,000 rpm for 10 min at 4°C. Then 25 ml of supernatant waslayered on top of 10 ml of 36% sucrose and centrifuged at 18,000 rpm for 80 minat 4°C in a Beckman SW28.1 rotor. The pellet was resuspended in 1.0 ml H2Oand microcentrifuged at 15,000 rpm for 30 min at 4°C. The pellet was resus-pended in 300 ml of H2O and then was layered on top of a CsCl discontinuousgradient (1.8 ml of 1.2 g/ml, 1.8 ml of 1.25 g/ml, and 1.3 ml of 1.3 g/ml). Thisgradient was centrifuged at 32,000 rpm and 15°C overnight in a Beckman SW50.1ultraclear rotor tube. Bands were side-pulled by using a 1-ml syringe with a22-gauge needle. The fractions were placed in separate 1.5-ml microcentrifugetubes, brought up to 1.5 ml with H2O, and then microcentrifuged at 15,000 rpmand 4°C for 30 min. The pellets were resuspended in 100 to 200 ml of 1 mM Tris(pH 9.0) and stored at 220°C.

Construction of recombinant plasmids. To construct in vitro expression vec-tors containing wild-type (G1) and mutant (A1) sequences of VV ORFs (A16L,E7R, and G9R), the specific DNA sequences were amplified from VV (WR)DNA template by PCR as described previously (16). The PCR-amplified prod-ucts were cloned into a plasmid vector (pTZ18R or pTZ19R; United StatesBiochemical) downstream of T7 RNA polymerase promoter sequences. PCRamplification primers were synthesized on an Applied Biosystems DNA synthe-sizer and purified by high-performance liquid chromatography (1). The se-quences of the PCR amplification primers were as follows: A16L-5*, 59-ACTGTGGATCCAGAATG(G/C)GGCAGCTGTTACTC-39; A16L-3*, 59-ATTTTGGATCCTTATCGTCTACGAACATTTATATC-39; E7R-5*, 59-ACTGTGGATCCAGAATGG(G/C)AACTGCTGCAACAATTC-39; E7R-3*, 59-ATTTTGGATCCTTATATATTTAGTTTATCTTTCATCAG-39; G9R-5*, 59-CTGCAGGTCGACTCTAGAATGG(G/C)TGGCGGAGTAAGTG-39; and G9R-3*, 59-CTGCAGGTCGACTCTAGATTAAATAGCTACAATTAGTATCCA-39. The PCRproducts were extracted with phenol-chloroform and concentrated by precipita-tion with ethanol. The PCR-amplified products and plasmid vectors were di-gested with either BamHI or XbaI, gel isolated, and ligated as follows: A16L(BamHI digested) into the BamHI site of pTZ19R, E7R (BamHI digested) intothe BamHI site of pTZ18R, and G9R (XbaI digested) into the XbaI site ofpTZ18R. DNA sequence analysis was used to confirm the authenticity of theplasmid constructions and identify the wild-type (G1) and the A1 mutants. Theconstruction of pL1 (G1) and pL1 (A1) have been detailed previously (16).

Production of antiserum. Polyclonal rabbit antisera to A16L, E7R, and L1Rproteins were generated by cloning coding regions of these ORFs into pET-16b(Novagen His-Tag expression vector system). This vector provides for high-stringency T7-lac promoter-inducible expression of an N-terminal fusion proteinto a cleavable His tag sequence for rapid affinity purification. Briefly, PCR-amplified DNA fragments consisting of the complete coding sequences of theE7R and L1R ORFs were ligated into the NdeI and BamHI sites of pET-16b. AnNdeI/BamHI fragment (;525 bp, encoding the C-terminal 171 amino acid res-idues of the A16L ORF) was isolated from a digestion of pTZ19R:A16L(G1)and ligated into the NdeI and BamHI sites of pET-16b. The host strain, Esche-richia coli BL21(DE3)(pLysS), was transformed with these recombinant vectors,and protein expression was induced with 1 mM isothiogalactopyranoside (IPTG)for 3 h. Cells were lysed by sonication and fractionated into soluble and insolublefractions. Fractions were separated by SDS-PAGE, and proteins were visualizedby staining with Coomassie brilliant blue. The fusion proteins induced frompET-16b:A16L and pET-16b:E7R were contained in the soluble fraction,whereas the fusion protein produced from the induction of pET-16b:L1R was inthe insoluble fraction of the cell extract. Both the A16L and E7R fusion proteinsmigrated as ;22-kDa proteins, and the L1R fusion protein migrated as an;29-kDa protein, as predicted. Antigen preparations for the immunization ofrabbits to produce antisera were isolated from the soluble fraction of the IPTG-induced cell extracts for A16L and E7R, whereas protein antigen from L1R-induced cultures was isolated from the bacterial inclusion bodies (35). Theprotein antigens were purified by immobilized metal affinity chromatography onNi21-nitrilotriacetic acid resin (Qiagen) according to the manufacturer’s instruc-tions. Antiserum to the G9R protein was produced from a synthetic multipleantigenic peptide consisting of residues 89 to 110 of the G9R ORF synthesizedon a tetravalent branching lysine core resin containing eight copies of the peptideper molecule. The protocol and schedule for immunization of the rabbits toproduce antisera have been detailed previously (16).

In vitro transcription and translation. Template plasmid DNAs were purifiedas directed by the manufacturer of the Riboprobe system (Promega). Plasmidswere linearized by digestion with restriction endonucleases as follows: EcoRI forpA16LG1, pA16LA1, pL1RG1, and pL1RA1, HindIII for pE7RG1 and pE7RA1,and SalI for pG9RG1 and pG9RA1. Linearized templates were transcribed in

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vitro by using T7 RNA polymerase. The mRNAs produced in the transcriptionreactions were translated in wheat germ extracts as directed by the manufacturer(Promega). Radiolabeling was performed with 0.5 mCi of L-[35S]methionine (1.153Ci/mmol; Amersham Corp.) per ml in 20 mM amino acids (minus methionine) orwith 2 mCi of [9,10-3H]myristic acid (54 Ci/mmol; Amersham) which had beendried under nitrogen and suspended by sonication in nuclease-free water per mlin 20 mM amino acids. Reaction mixtures (15 ml) were incubated at 25°C for 60min. The radioactive polypeptides were analyzed on SDS–10% polyacrylamidegels and visualized by fluorography (4) on Kodak XAR-5 film at 270°C.

RESULTS

Time course radiolabel of VV myristylproteins. During ourinitial studies on the VV L1R protein, we noted that severalother proteins labeled with [3H]myristic acid during a VVinfection (17). Given that viral myristylproteins are often in-volved in assembly or, in rare instances, DNA replication (9),identification and characterization of VV myristylproteinsseemed to be an important approach to understanding of theseprocesses in the VV life cycle. Virus-infected cells were radio-labeled with [3H]myristic acid at various times p.i. to examinethe kinetics of myristylprotein expression. VV early proteinsare synthesized prior to DNA synthesis, within the first 4 h ofinfection (37). Beginning at about 4 h p.i., late protein expres-sion commences and early gene expression is attenuated. Ascan be seen in Fig. 1, five VV proteins with apparent molecularmasses of 92, 39, 36, 25, and 17 kDa labeled with myristic acidat 4 h p.i. These increase in intensity with time, as do a numberof other bands. Whether these other bands represent otheracylproteins, breakdown products, or reutilization of label isnot known. In any case, further studies were continued only onthe five major species indicated. In addition, an early 14-kDaprotein is radiolabeled with myristic acid as well. This proteinremains to be identified. All of the late myristylproteins exceptthe 92-kDa protein are expressed by both the WR and CHstrains of VV, suggesting they are virally encoded (Fig. 2).Based on earlier work, the 25-kDa protein was identified asthat expressed by the L1R ORF. We also identified the 92-kDaprotein as the VV homolog to the cowpox virus A-type inclu-sion protein (34a). This left the identities of the 39-, 36-, and17-kDa proteins to be determined.

Identities of the late myristylproteins. The entire genome ofthe CH strain of VV has been sequenced (20). The predictedamino acid sequences of all of the VV ORFs were examinedfor the presence of the N-myristylation consensus sequence.Three proteins were identified by this search: products of theG9R, A16L, and E7R ORFs (Table 1). The sequence sur-rounding the translational start site of each ORF (TAAATG)is characteristic of a late gene promoter (11). In addition, noneof the three ORFs has an early transcription termination se-quence. The predicted molecular masses of the proteins are38.8 kDa (G9R), 43.6 kDa (A16L), and 19.5 kDa (E7R). Allthree have an N-terminal sequence consisting of MGXXX(S/T), which adheres to the N-myristylation consensus sequence(13). Like L1R, the proteins encoded by A16L and G9R areboth predicted to have a hydrophobic carboxy terminus.

In vitro transcription and translation. To confirm that theG9R, A16L, and E7R ORFs do in fact encode the late myri-stylproteins observed in the in vivo studies, an in vitro ap-proach was used. A number of investigators have studied myri-stylation in vitro by using either rabbit reticulocyte or wheatgerm lysates (8, 12, 23). These studies have been successfulbecause both lysates contain N-myristyltransferase activity.Previous studies using the wheat germ translation system dem-onstrated that the VV 25-kDa myristylprotein was expressedby the L1R ORF (16). The same system was used to demon-strate the ability of proteins encoded by G9R, A16L, and E7Rto be myristylated in vitro. To achieve this, each of the ORFswas PCR amplified from a VV DNA genomic template andcloned into an expression vector plasmid behind a T7 pro-moter. Three additional plasmid constructs were designed such

FIG. 1. Time course label of myristylated VV proteins. BSC-40 cells infectedwith the WR strain of VV (MOI of 10 PFU per cell) were radiolabeled with [3H]myristic acid during 4-h intervals at various times p.i. Cells were harvested, andproteins were separated on a 10% tricine polyacrylamide gel. The gel wasfluorographed by using 22.2% PPO in DMSO, dried, and exposed to KodakBiomax MR film at 270°C. The migration of molecular weight markers (myosin[200 kDa], phosphorylase b [97.5 kDa], bovine serum albumin [69 kDa], ovalbu-min [46 kDa], carbonic anhydrase [30 kDa], and trypsin inhibitor [21.5 kDa]) isdepicted on the left. Lanes (left to right): 0 to 4, 4 to 8, 8 to 12, 12 to 16, and 16to 24 h p.i. Arrows indicate the major late myristylproteins.

FIG. 2. Comparison of WR and CH myristylated proteins. BSC-40 cells wereinfected with either the WR or CH strain of VV (MOI of 10 PFU per cell) andradiolabeled with [3H]myristic acid from 3.5 to 10 h p.i. One plate of uninfectedBSC-40 cells (lane MI) was also radiolabeled. Cells were harvested, and proteinswere resolved by SDS-PAGE, using a 12% polyacrylamide gel. The gel wasfluorographed by using 22.2% PPO in DMSO, dried, and exposed to KodakXAR-5 film at 270°C. Migration of molecular weight markers, in kilodaltons, isindicated at the left.

TABLE 1. ORFs containing the consensus sequenceat the initiation sitea

ORF Size (kDa)b N-terminal sequence

A16L 43.6 (36) MGAAVTLNRIKIAG9R 38.8 (39) MGGGVSVELPKRDL1R 27.5 (25) MGAAASIQTTVNTE7R 19.5 (17) MGTAATIQTPTKL

a All ORFs are of the late temporal class. All except E7R exhibit the proposedmembrane-associated region at the C terminus.

b Numbers in parentheses indicate the apparent molecular masses of theproteins as determined by SDS-PAGE mobility. Numbers not in parenthesesindicate predicted molecular mass.

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that the glycine adjacent to the initiating methionine was mu-tated to an alanine. It has been well established that mutatingthe penultimate glycine of an N-myristylated protein to analanine is sufficient to inhibit myristylation (53). If these are thegenes encoding the three unidentified late myristylproteins,then transcription and translation of the cloned wild-typegenes should result in proteins that can be labeled with myristicacid, whereas the gene products of the cloned mutated geneswill be blocked in their ability to be myristylated.

G9, G9A1, A16, A16A1, E7, E7A1, L1, and L1A1 mRNAswere synthesized in vitro by transcription with T7 RNA poly-merase. The mRNA was used to program wheat germ extractslabeled with either [35S]methionine or [3H]myristic acid, andthe translation products were analyzed by SDS-PAGE. In [35S]methionine-labeled extracts, each mRNA served as the tem-plate for a single protein band of appropriate molecular size,with the exception of the A16 and A16A1 mRNAs (Fig. 3A).These two messages produced the expected 36-kDa band andseveral additional protein bands. The band migrating at ap-proximately 65 kDa may represent either a dimer or an mRNAtranscript that did not terminate at the transcriptional stop site.The bands migrating below 36 kDa are likely caused by nicks inthe mRNA or by the translational machinery falling off thetemplate prematurely. In contrast, bands in the [3H]myristicacid-labeled extracts were present only in those which hadmRNA from wild-type DNA (Fig. 3B). None of the A1 mu-tants produced translation products that labeled with myristicacid, despite the ability of the respective mRNAs to producepolypeptides of the expected size, as shown by incorporation of[35S]methionine. Taken together, these experiments demon-strate that not only are the proteins encoded by the G9R,A16L, and E7R ORFs myristylated, but the penultimate gly-cine residue is the apparent target site for myristylation. Fur-thermore, this finding provides support for the conclusion thatthe three previously unidentified 38-, 36-, and 17-kDa myristyl-proteins are encoded by these ORFs.

Transmembrane prediction plots. Because many myristy-lated viral proteins are targeted to cellular membranes orviral envelopes, we were interested in seeing if the proteinsencoded by the G9R, A16L, and E7R ORFs were predictedto contain transmembrane domains. To this end, the aminoacid sequence of each protein was analyzed by using theTMpred program available on the World Wide Web at http://ulrec3.unil.ch/software/TMPRED_form.html (24a). Thisprogram makes predictions about helix regions with a lengthbetween 17 and 33 amino acids and gives a weighted score

for each region found. In addition, the program makes pre-dictions about the possible membrane topology, suggestingwhether the N terminus lies inside or outside the membrane.Hydropathy plots are shown for each protein (Fig. 4), asprovided by the TMpred program. The TMpred analysispredicts that G9R has one transmembrane domain betweenamino acids 322 and 340 and that A16L has one transmem-brane domain between amino acids 342 and 360. In com-parison, L1R is predicted to have a transmembrane domainbetween amino acids 186 and 204. The topology predictionfor G9R is that the N terminus is inside, whereas the pre-dictions for A16L and L1R place each N terminus outsidethe membrane. E7R is not predicted to have a transmem-brane domain.

Subcellular fractionation of VV-infected cells. Because G9Rand A16L were both predicted to have a membrane-associatedC terminus, subcellular fractionation studies were performedto determine if, like L1R, these two proteins are associatedwith membranes in vivo. Furthermore, since E7R was notpredicted to have a membrane-associated domain, we wantedto determine whether it was a soluble protein. We used aprocedure that separates VV-infected cells into the TCE, NP,PNS, P15, P100, and S100 fractions. All fractions obtainedrepresented equal portions of the total cell lysate that washarvested. The NP contained the nuclear membrane and per-haps portions of the endoplasmic reticulum (ER), while theP15 and P100 fractions contained the plasma membrane, por-tions of the ER, and the trans-Golgi network. The PNS andS100 fractions contained cytosol material, including solubleand non-membrane-associated proteins. Equal volumes ofeach fraction were analyzed by SDS-PAGE. Proteins on the gelwere electroblotted to nitrocellulose and then probed with apolyclonal antiserum directed against L1R, G9R, A16L, orE7R.

As expected, L1R was found only in the TCE and NP frac-tions (Fig. 5A). Since it has already been established that L1Ris associated with the inner envelope of mature virions (42), itshould have been found only in the fractions containing theinsoluble membrane-associated proteins. Surprisingly, A16Ldid not colocalize to the same fractions as L1R (Fig. 5B).Although both A16L and L1R have a predicted membrane-associated domain at their C termini, A16L was found only inthe soluble fractions (TCE, PNS, and S100). E7R was alsofound only in the TCE, PNS, and S100 fractions (Fig. 5C),suggesting that it is also a soluble protein. Interestingly, theanti-E7R antiserum reacts with two proteins that migrate closetogether. Perhaps this is caused by disulfide bonds between twomolecules of E7R as is seen with L1R (57). Alternatively, oneof the bands may be an unrelated cross-reactive protein. G9Rwas detected only in the TCE, not in any of the subcellularfractions (Fig. 5D). This finding suggests either that G9R ismade in much lower quantities than the other myristylproteinsor that the G9R protein is labile. It is also possible that theantiserum against G9R is not sensitive enough for this assay.Although the localization of G9R remains unknown, it appearsthat neither E7R nor A16L is membrane associated.

Packaging in mature virions. Previous studies have shownthat the VV L1R protein is a component of both IMV andEEV (42). These are the two major forms of infectious virionsproduced during infection. One feature that distinguishes IMVfrom EEV is the number of membranes that surrounds thevirus particle. IMV has a double membrane acquired from thecisternae of the intermediate compartment between the Golgicomplex and the ER (48). EEV particles are wrapped by ad-ditional layers of membranes derived from the trans-Golgi orearly endosomal networks (47, 52). This difference in the num-

FIG. 3. In vitro synthesis and myristylation of wild-type and mutant G9R,A16L, L1R, and E7R proteins. Wheat germ extracts were programmed withmRNA (as indicated above the lanes) transcribed in vitro with T7 RNA poly-merase. Translation reactions were labeled with [35S]methionine (A) or [3H]myristic acid (B). Migration of molecular weight markers, in kilodaltons, isindicated at the left.



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FIG. 4. Hydropathy plots of the VV major late myristylproteins L1R (A), A16L (B), E7R (C), and G9R (D). The plots were generated by using the Hopp andWoods algorithm (45). Positive values (above the center line) indicate hydrophobicity; negative values indicate hydrophilicity.



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FIG. 4—Continued.



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ber of lipid layers surrounding the virus particles gives IMVand EEV separate and distinct relative buoyant densities inisopycnic cesium chloride gradients. Furthermore, the proteinprofile of EEV is not identical to that of IMV, such that thereare EEV-specific proteins (2, 3, 14, 15, 24, 38–40, 56) andIMV-specific proteins (40, 54). However, many proteins iso-lated from virus particles, like L1R, are found in both IMV andEEV.

To determine whether the late myristylproteins were con-tained within mature virions, IMV and EEV were purifiedfrom RK13 cells infected with the IHD-J strain of VV. Proteinsfrom purified virus were resolved by discontinuous SDS-PAGEand identified by immunoblot analysis using specific polyclonalantisera. A sample of the TCE was reserved prior to viruspurification to confirm expression of each myristylprotein. Todemonstrate the purity of each virus preparation, IMV andEEV were assayed for the presence of P37, an EEV-specificprotein (24). As expected, P37 is present in purified EEV andis absent in IMV (Fig. 6A). In addition, both virus preparationswere probed with the antiserum against L1R to demonstratethat equal concentrations of IMV and EEV were being loadedon the polyacrylamide gels (Fig. 6B). The observed results forG9R, E7R, and A16L were surprising. G9R was observed atsuch a low level as to make it barely detectable (data notshown). E7R, on the other hand, was observed primarily in theTCE and EEV but was present as only a very faint dimer inIMV (Fig. 6C). Last, A16L was detected only in the TCE andwas completely absent from both forms of mature virions (Fig.6D). Although A16L is soluble, it was surprising to find that itis not assembled into virions during maturation, especiallysince it is a late protein.

DISCUSSION

In order for a virus to infect a cell, a number of obstaclesmust be overcome. Many of these are associated with entryinto the cell, replication of the genome, and expression ofviral proteins. Yet it is also critical that viral proteins, onceexpressed, get to the correct location in the cell at the appro-priate time in order for assembly, maturation, and egress ofinfectious progeny virions to occur. Posttranslational modifi-cations, such as myristylation, provide a means for many vi-ruses to successfully complete their infectious cycle. Inhibitionof myristylation is, in many cases, sufficient to prevent forma-tion or release of infectious progeny virions. This has beendemonstrated for a number of viruses, including picornavirus(31, 34), Sindbis virus (18), polyomavirus, and simian virus 40(32, 51), human immunodeficiency virus (5, 19, 21), and vac-cinia virus (17, 43).

The mechanisms by which VV progeny virions assemble,mature, and exit the cell are just beginning to be understood.Approximately 10% of VV proteins, including at least five latemyristylproteins and six late palmitylated proteins (7), are ei-ther myristylated or palmitylated. This finding suggests thatacylated proteins may play important roles during late eventsin the infectious cycle. This is already proving to be the case fortwo acylated proteins, L1R and P37. L1R, a late myristylpro-tein, appears to play a key role in morphogenesis, since onlyimmature, noninfectious particles are formed when L1R is notexpressed (43). P37 is a palmitylated late protein, which hasbeen shown to be essential for egress of enveloped virions (22,24). Study of other late VV acylproteins, such as A16L, G9R,and E7R, should provide insight into the mechanisms involvedin assembly, morphogenesis, and egress of infectious progenyvirions.

FIG. 5. Subcellular localization of VV late myristylproteins. BSC-40 cellswere infected with the IHD-J strain of VV at an MOI of 10 PFU/cell. Infectedcells were harvested 24 h p.i. and pelleted by centrifugation. The pellet wasresuspended in hypotonic buffer and Dounce homogenized. An aliquot wasreserved as the TCE, and the remainder was centrifuged at 1,300 rpm. The NPwas resuspended in hypotonic buffer, and the PNS was reserved. The resus-pended NP was microcentrifuged, and the pellet (P15) was resuspended inhypotonic buffer. The supernatant was centrifuged at 33,000 rpm to separate theP100 and S100 fractions. See Materials and Methods for details on samplepreparation. A portion of each fraction was run on SDS–12% polyacrylamidegels, transferred to nitrocellulose, and then probed with polyclonal antiseraagainst L1R (A), A16L (B), E7R (C), and G9R (D). Sizes of markers areindicated in kilodaltons on the left.

FIG. 6. IMV or EEV localization of myristylated VV proteins. RK13 cellswere infected with the IHD-J strain of VV at an MOI of 5 PFU/cell. Infectedcells were harvested at 24 h p.i. and sonicated briefly. An aliquot was reserved asthe TCE. Total virus was first pelleted through a sucrose cushion and then separatedinto IMV and EEV fractions, using a CsCl discontinuous gradient. For details, seeMaterials and Methods. Aliquots of purified IMV and EEV were run on SDS–12% polyacrylamide gels, and electroblotted to nitrocellulose, and probed withpolyclonal antisera against P37 (A), L1R (B), E7R (C), and A16L (D).



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In vitro expression of the A16L, G9R, and E7R ORFs dem-onstrated that the penultimate glycine is the target site foraddition of myristic acid. Further, the first five amino acidshave homology with the N-myristylation consensus sequence.Together, these findings indicate that all three follow the nor-mal pattern of N-terminal myristylation. To begin to charac-terize these three proteins, it was important to determinewhether they associate with membranes or remain soluble inthe cytoplasm. Membrane association would suggest a role inmorphogenesis or egress, while solubility might indicate anassociation with another protein and perhaps a role in assem-bly of the viral core. Both A16L and E7R were found only inthe soluble subcellular fractions, providing strong evidencethat neither protein is associated with membranes. The solu-bility of G9R remains unclear, since it could be detected onlyin the TCE. Its hydropathy profile is very similar to those ofA16L and E7R, suggesting that it might also be soluble. Giventhe enveloped nature of VV, it is somewhat surprising that atleast two, and maybe three, of the late myristylproteins are notassociated with membranes.

Because most myristylated viral proteins are components ofthe capsid or viral envelope, it was of interest to determine ifthis was also the case for A16L, G9R, and E7R. By probingpurified virions with specific polyclonal antisera, it was deter-mined that E7R (and perhaps G9R) resides within matureinfectious virions. The fate of A16L is less clear. Surprisingly,this protein was not detected in purified virions, although itwas seemingly abundant in the TCE and in soluble fractions ofinfected cells. There remains much to learn about the functionsof A16L, G9R, and E7R. Studies need to be performed todetermine if these proteins are essential to the virus for repli-cation in order to begin to understand their functions. Further,in vivo studies of nonmyristylating mutants would be of interestto determine the role of myristylation for each protein.

ACKNOWLEDGMENTS

We thank the staff at CSL for oligonucleotide preparation and DNAsequencing services. We also thank our colleagues for providing ideasand critical evaluations of this work.

This research was funded by NIH grant AI-21335.

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Identification and Analysis of Three Myristylated Vaccinia Virus Late